Iuliana Popa

Characterization of the ceramide moieties of sphingoglycolipids from mouse brain by ESI-MS/MS identification of ceramides containing sphingadienine

Sphingoglycolipids (SGLs) are cell membrane constituents. As the ceramide structure influences the biological properties of the SGL, we characterized by electrospray ionization tandem mass spectrometry the molecular species of ceramides present in SGL of mouse brain. We report here for the first time the presence in mammalian brain of sphingadienine (d18:2). Sphingenine (d18:1) is present in all SGL species, in contrast to eicosasphingenine (d20:1), which is a constituent of only gangliosides. Sphingadienine is present in galactosylceramide and sulfatides. Free ceramides contain the three types of bases. Thus, there could be two separate pools of free ceramides (d18:1, d18:2 and d20:1, d18:1) as precursors of complex SGL.

Endogenous retroviral genes, Herpesviruses and gender in Multiple Sclerosis.

Unexpected findings on endogenous retroviral elements expressed in cells from patients with Multiple Sclerosis appear to open a new avenue of research, after years of research dedicated to the understanding of their biological significance in human health and disease. Human endogenous retroviral family W (HERV-W) RNA present in circulating viral particles (Multiple Sclerosis associated RetroViral element, MSRV) has been associated with the evolution and prognosis of Multiple Sclerosis. HERV-W elements encode a powerful immunopathogenic envelope protein (ENV) that activates a pro-inflammatory and autoimmune cascade through interaction with Toll-Like Receptor 4 (TLR4) on antigen-presenting cells, and triggers superantigen-like dysregulation of T-lymphocytes. HERV-W/ENV antigen has further been shown to be an upstream inducer of immunopathogenicity like that in MS and has repeatedly been detected in association with MS lesions in post-mortem brain studies. ENV protein now represents a novel target in MS, in our ongoing development of a neutralising therapeutic antibody. We here review the pieces of a puzzle, which now offer a consistent picture for Multiple Sclerosis aetiopathogenesis. Interestingly, at the gene-environment interface, this picture also includes gender-related specificities through the potential interplay with endogenous retrovirus type W copies present on the X chromosome.

The lipid alterations in the stratum corneum of dogs with atopic dermatitis are alleviated by topical application of a sphingolipid-containing emulsion

Background.  Atopic dermatitis (AD) results from an altered skin barrier associated with defects in the lipid composition of the skin. Dogs with AD present similar clinical symptoms to humans, and may be a useful model for investigations into AD.

Analysis of free and protein-bound ceramides by tape stripping of stratum corneum from dogs

The free and protein-bound ceramides of dog stratum corneum (SC) were analyzed by thin-layer chromatography after tape stripping of the abdomen of five dogs. The sphingoid bases were identified by gas–liquid chromatography as sphingosine, phytosphingosine, and 6-hydroxysphingosine. Electrospray ionization-ion trap mass spectrometry was used to characterize the protein-bound ceramides containing sphingosine and omega-hydroxy long-chain fatty acids. Although the molecular species were the same ones in all dogs, wide quantitative variations in the patterns of SC ceramides were observed in different breeds of dogs. The free ceramide concentration changed with the depth of SC, with a higher concentration in the deep layers, whereas the concentration of protein-bound ceramides remained constant. These results show that canine SC is close to that of humans with respect to ceramides.

Different mechanisms are involved in apoptosis induced by melanoma gangliosides on human monocyte-derived dendritic cells

Tumor escape is linked to multiple mechanisms, notably the liberation, by tumor cells, of soluble factors that inhibit the function of dendritic cells (DC). We have shown that melanoma gangliosides impair DC differentiation and induce their apoptosis. The present study was aimed to give insight into the mechanisms involved. DC apoptosis was independent of the catabolism of gangliosides since lactosylceramide did not induce cell death. Apoptosis induced by GM3 and GD3 gangliosides was not blocked by inhibitors of de novo ceramide biosynthesis, whereas the acid sphingomyelinase inhibitor desipramine only prevented apoptosis induced by GM3. Furthermore, our results suggest that DC apoptosis was triggered via caspase activation, and it was ROS dependent with GD3 ganglioside, suggesting that GM3 and GD3 induced apoptosis through different mechanisms.

A new approach for drug discovery from glycobiology and phage-displayed peptide library technology

Peptides which mimic functional activities of glycosphingolipids were prepared by a technology of phage-displayed peptide library using monoclonal antibodies against glycosphingolipids. These peptides were named glyco-replica peptides. Peptides prepared with anti-GD1alpha antibody by this technology were found to contain WHW as common motif, and they showed suppressive activity not only on adhesion between hepatic sinusoidal endothelial cells and lymphosarcoma RAW117-H10 cells, but also on metastasis of the tumor cell to the liver and lung. The WHW motif seems to be important to mimic the functional activity of the ganglioside GD1alpha. Next, we prepared GD3-replica peptides using a monoclonal antibody against GD3 (4F6). A peptide, GD3-P4 with highest affinity to 4F6 was used to immunize mice to examine if the mice show their immune response to raise antibodies against GD3. We confirmed the immune response and succeeded in the production of a monoclonal antibody (3D2) against GD3. The monoclonal antibody 3D2 showed specific binding to GD3 on a thin-layer chromatography plate and also melanoma tissues. Interestingly, the amino acid sequence of the CDR regions of light and heavy chains showed high similarity with those of the original GD3 monoclonal antibody (4F6) used for the preparation of GD3-replica peptide. The technology of the phage-displayed peptide library was applied to in vivo bio-panning study using an angiogenesis experimental model. The obtained peptides were found to show strong binding property to the neo-vasculature system and to be quite useful to carry an anti-tumor drug to the tumor tissue. Based on these experimental results, we discuss about some applications of this method to drug discovery.

Studies of compounds that enhance sphingolipid metabolism in human keratinocytes.

Several products are known to inhibit the biosynthesis of ceramides and glucosylceramides, but very few stimulate this process. We studied the influence of a hydrolysate of potato proteins (Lipidessence®) in vitro on the sphingolipid metabolism of normal human epidermal keratinocytes. By measuring growth with the thymidine uptake assay, it was seen that Lipidessence®, added in the culture medium up to an 8% concentration, did not change significantly the proliferation rate of keratinocytes, but beyond this concentration a progressive dose‐dependent inhibition of growth was noticeable. Following incubation of cells with the product at 5% and 10% concentrations for 2 days, the lipids were extracted. The different lipid classes were separated by fractionation on columns of aminopropyl silica gel and analyzed by high‐performance thin‐layer chromatography. When keratinocytes were cultivated in the presence of Lipidessence®, the biosynthesis of cholesterol, phosphatidylcholine, phosphatidylserine and gangliosides was stimulated, and a major increase was noticeable in the biosynthesis of free fatty acids, free ceramides, glucosylceramide and sphingomyelin. Radioactive [14C]‐serine was used as a precursor of sphingoid bases to study sphingolipid biosynthesis. After migration of lipid fractions on thin‐layer plates, autoradiography showed that free ceramides and glucosylceramide were labeled, thus suggesting that de novo biosynthesis was accounting for the increased cellular content in sphingolipids.

Regulation of SREBPs by Sphingomyelin in Adipocytes via a Caveolin and Ras-ERK-MAPK-CREB Signaling Pathway

Sterol response element binding protein (SREBP) is a key transcription factor in insulin and glucose metabolism. We previously demonstrated that elevated levels of membrane sphingomyelin (SM) were related to peroxisome proliferator–activated receptor-γ (PPARγ), which is a known target gene of SREBP-1 in adipocytes. However, the role of SM in SREBP expression in adipocytes remains unknown. In human abdominal adipose tissue from obese women with various concentrations of fasting plasma insulin, SREBP-1 proteins decreased in parallel with increases in membrane SM levels. An inverse correlation was found between the membrane SM content and the levels of SREBP-1c/ERK/Ras/PPARγ/CREB proteins. For the first time, we demonstrate the effects of SM and its signaling pathway in 3T3-F442A adipocytes. These cells were enriched or unenriched with SM in a range of concentrations similar to those observed in obese subjects by adding exogenous natural SMs (having different acyl chain lengths) or by inhibiting neutral sphingomyelinase. SM accumulated in caveolae of the plasma membrane within 24 h and then in the intracellular space. SM enrichment decreased SREBP-1 through the inhibition of extracellular signal-regulated protein kinase (ERK) but not JNK or p38 mitogen-activated protein kinase (MAPK). Ras/Raf-1/MEK1/2 and KSR proteins, which are upstream mediators of ERK, were down-regulated, whereas SREBP-2/caveolin and cholesterol were up-regulated. In SM-unmodulated adipocytes treated with DL-1-Phenyl-2-Palmitoylamino-3-morpholino-1-propanol (PPMP), where the ceramide level increased, the expression levels of SREBPs and ERK were modulated in an opposite direction relative to the SM-enriched cells. SM inhibited the insulin-induced expression of SREBP-1. Rosiglitazone, which is an anti-diabetic agent and potent activator of PPARγ, reversed the effects of SM on SREBP-1, PPARγ and CREB. Taken together, these findings provide novel insights indicating that excess membrane SM might be critical for regulating SREBPs in adipocytes via a MAPK-dependent pathway.

The weak rate of sphingolipid biosynthesis shown by basal keratinocytes isolated from aged vs. young donors is fully rejuvenated after treatment with peptides of a potato hydrolysate

A new study was carried out to bring more information on the effect of the potato proteins ferment. Basal keratinocytes obtained from freshly excised skin samples of two groups of five donors, a young one (25–36‐year‐old) and an aged one (59–70‐year‐old) were established in culture. The results showed a downward trend in the content of all lipid fractions in untreated keratinocytes of aged donors when compared with young ones. We found major differences in the response of keratinocytes to potato proteins ferment treatment between young and old donors. Whereas the lipid content of cells from young donors increased either moderately or actually decreased in some cases in comparison with the untreated controls, the lipid biosynthesis was strongly stimulated in aged donors’ keratinocytes whose lipid contents globally became close to those found in young donors. However, the changes elicited by potato proteins ferment treatment were not seen at the same extent for all lipid classes. Cholesterol content increased up to three‐fold and alpha‐hydroxy fatty acids were augmented up to seven‐fold, whereas the increase in normal fatty acids was quite moderate. In sphingolipids labelled by incubation of keratinocytes in culture medium containing [14C]‐serine, ceramides and glucosylceramides in cells from aged donors showed the highest uptake of radioactivity, with somewhat less incorporation in sphingomyelin and gangliosides. Therefore, it seems that potato proteins ferment has a much more potent stimulatory activity on the lipid biosynthesis of basal keratinocytes of aged donors, thereby normalizing the cellular lipid content that obviously decreases along with ageing. Although our results were obtained only with basal keratinocytes in this study, potato proteins ferment could be beneficial to maintain an efficient skin barrier in ageing people, provided that the peptides can get through to the basal membrane upon topical application.

Production of multiple brain-like ganglioside species is dispensable for fas-induced apoptosis of lymphoid cells.

Activation of an acid sphingomyelinase (aSMase) leading to a biosynthesis of GD3 disialoganglioside has been associated with Fas-induced apoptosis of lymphoid cells. The present study was undertaken to clarify the role of this enzyme in the generation of gangliosides during apoptosis triggered by Fas ligation. The issue was addressed by using aSMase-deficient and aSMase-corrected cell lines derived from Niemann-Pick disease (NPD) patients. Fas cross-linking elicited a rapid production of large amounts of complex a- and b-series species of gangliosides with a pattern and a chromatographic behavior as single bands reminiscent of brain gangliosides. The gangliosides were synthesized within the first ten minutes and completely disappeared within thirty minutes after stimulation. Noteworthy is the observation that GD3 was not the only ganglioside produced. The production of gangliosides and the onset of apoptotic hallmarks occurred similarly in both aSMase-deficient and aSMase-corrected NPD lymphoid cells, indicating that aSMase activation is not accountable for ganglioside generation. Hampering ganglioside production by inhibiting the key enzyme glucosylceramide synthase did not abrogate the apoptotic process. In addition, GM3 synthase-deficient lymphoid cells underwent Fas-induced apoptosis, suggesting that gangliosides are unlikely to play an indispensable role in transducing Fas-induced apoptosis of lymphoid cells.